DNA constructs are typically propagated as plasmids. Plasmids are frequently constructed by cloning a first polynucleotide sequence into a vector. The vector generally comprises sequences required for propagation in at least one host cell, but it often also comprises sequences that contribute to the functioning of the first polynucleotide sequence. For example a vector may comprise elements that affect the expression of a polypeptide encoded by the first polynucleotide sequence such as promoters, enhancers, introns, terminators, translational initiation signals, polyadenylation signals, replication elements, RNA processing and export elements, and elements that affect chromatin structure that become operably linked to the first polynucleotide. The process of optimizing a polynucleotide for a specific function often comprises creating a plurality of polynucleotides, cloning them into the same vector to create a first plurality of cloned polynucleotides and measuring a property of some of the cloned polynucleotides.
Because the process of cloning polynucleotides into a single vector is relatively simple, while the process of constructing a vector is more complex and costly, optimization almost always focuses on creating variation in the cloned polynucleotide and very rarely on variations in the vector. Even if the vector sequence is varied, this will typically be done by selecting from a small number of pre-existing vectors rather than by deliberately constructing a new set of vectors. However vectors frequently contain many or even most of the elements that determine the function of the cloned polynucleotide, for example the expression of the polynucleotide in an expression-host. The functional performance of many of these elements may depend on the precise host cell being used, for example some elements that perform well in human cells may perform poorly in rodent cells, the same vector is often used in both.
Furthermore, many available vectors have been constructed by standard restriction site cloning methods and derived from other vectors wherein the functional elements have not been well defined. Consequently many vectors contain “fossil” sequences that are unnecessary for their function but have just been included because of imprecise cloning methods or a lack of understanding of function (for example the fl phage origin of replication, originally incorporated for generation of phagemids which can be found in many vectors that are never used to make phagemids), or they contain sequences that actually compromise function (for example the use of the beta lactamase gene as a selectable marker which exacerbates instability in vectors such as lentiviruses).
Because of the immense size of sequence space, there is no effective way to test all possible permutations of a polymeric biological molecule such as a nucleic acid or protein for a desired property. To test each possible nucleotide base at each position in a vector, rapidly leads to such a large number of molecules to be tested such that no available methods of synthesis or testing are feasible, even for a polymer of modest length. Furthermore, most molecules generated in such a way would lack any measurable level of the desired property. Total sequence space is very large and the functional solutions in this space are sparsely distributed.
There is thus a need in the art for methods to efficiently identify vector components that contribute to performance, and to assess this performance.
Typical methods for introducing DNA into a cell include DNA condensing reagents such as calcium phosphate, polyethylene glycol, lipid-containing reagents such as liposomes, multi-lamellar vesicles as well as virus-mediated strategies. However, such methods can have certain limitations. For example, there are size constraints associated with DNA condensing reagents and virus-mediated strategies. Further, the amount of nucleic acid that can be transfected into a cell is limited in viral strategies. In addition, not all methods facilitate insertion of the delivered nucleic acid into cellular nucleic acid, and while DNA condensing methods and lipid-containing reagents are relatively easy to prepare, the insertion of nucleic acid into viral vectors can be labor intensive. Virus-mediated strategies can be cell-type specific or tissue-type specific, and the use of virus-mediated strategies can create immunologic problems when used in vivo.
Integration of heterologous DNA into a target genome, and the expression levels of genes encoded by the integrated heterologous DNA can be increased by the configuration of DNA elements. The efficiency of integration, the size of the heterologous DNA sequence that can be integrated, and the number of copies of the heterologous DNA sequence that are integrated into each genome can often be further improved by using transposons. Transposons or transposable elements include a short nucleic acid sequence with terminal repeat sequences upstream and downstream. Active transposons can encode enzymes that facilitate the excision and insertion of the nucleic acid into target DNA sequences. A number of transposable elements have been described in the art that facilitate insertion of nucleic acids into the genome of vertebrates. For example, transposable elements discovered from various sources, for example, an engineered transposon from the genome of salmonid fish called sleeping beauty; piggyBac transposon from lepidopteran cells; piggyBac transposon from the bat Myosis lucifugus; mariner transposon first discovered in Drosophila and; an engineered transposon and transposon inverted repeats from the frog species, Rana pipiens called frog prince.
Different transposable elements show different preferences for the genomic sites at which they integrate. For example the piggyBac and piggyBat transposons have a preference for transcriptionally inactive regions. Although this may be an advantage for the “wild” transposon which does not wish to disrupt gene expression in its host and risk killing it, it is a disadvantage for transposons that are being used to maximize gene expression. Thus although a number of transposable elements capable of facilitating insertion of nucleic acids into the eukaryotic genome have been identified in the art, there exists a need for alternative transposable elements and enhanced constructs that facilitate higher expression levels from inserted DNA, either because of higher insertion efficiency or because the genomic insertions are made at more favorable positions within the genome, compared with transposable elements currently described in the art.